title: Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target.
abstract: Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))•(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu•Py sequences may be pertinent to gene regulation.
authors.affiliation: Nucleic Acids Research Laboratory, Department of Chemistry, University of Delhi (North Campus), Delhi 110007, India.
authors.forename: Shikha
authors.initials: S
authors.lastname: Kaushik
authors.forename: Mahima
authors.initials: M
authors.lastname: Kaushik
authors.forename: Fedor
authors.initials: F
authors.lastname: Svinarchuk
authors.forename: Claude
authors.initials: C
authors.lastname: Malvy
authors.forename: Serge
authors.initials: S
authors.lastname: Fermandjian
authors.forename: Shrikant
authors.initials: S
authors.lastname: Kukreti
mesh_terms: D002413:Cations, Divalent / Q000737:chemistry / Q000528:radiation effects; D004247:DNA / Q000737:chemistry* / Q000528:radiation effects; D018390:Gene Targeting / Q000379:methods*; D016758:Genes, jun / Q000528:radiation effects; D006358:Hot Temperature; D006801:Humans; D008274:Magnesium / Q000737:chemistry / Q000528:radiation effects; D009690:Nucleic Acid Conformation / Q000528:radiation effects; D009691:Nucleic Acid Denaturation / Q000528:radiation effects; D011685:Purine Nucleotides / Q000737:chemistry* / Q000528:radiation effects; D011742:Pyrimidine Nucleotides / Q000737:chemistry / Q000528:radiation effects; D012964:Sodium / Q000737:chemistry / Q000528:radiation effects; D014466:Ultraviolet Rays
chemical_list: D002413:Cations, Divalent; D011685:Purine Nucleotides; D011742:Pyrimidine Nucleotides; C051802:triplex DNA; D004247:DNA; D012964:Sodium; D008274:Magnesium